Matrix interferences evaluation employing GC and LC ... · PDF fileMatrix interferences...
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Matrix interferences evaluation employing GC and LC coupled to triple quadrupole tandem mass spectrometry Uclés S. 1 , Lozano A. 1 , Sosa A. 2 , Amadeo R. Fernández-Alba 1 1 Agrifood Campus of International Excellence (ceiA3), European Union Reference Laboratory for Pesticide Residues in Fruits and Vegetables. University of Almería (Spain); e-mail: [email protected] 2 National Agricultural Technology Institute (INTA). Concordia Agricultural Experimental Station (Argentina) INTRODUCTION Gas and liquid chromatography coupled to triple quadrupole tandem mass spectrometry are two very powerful tools commonly employed for the analysis of pesticide residues in fruit and vegetables. However, due to the big quantity and complexity of the commodities different responses are produced, making difficult to identify/quantify a compound. To get a clear evaluation twenty five different matrices were purchased from local markets in Almería, extracted with four commonly used extraction methods (citrate QuEChERS with/without clean-up, ethyl acetate extraction method [1], and Dutch mini-Luke method [2]), and evaluated with both GC-QqQ-MS/MS and LC-QqQ-MS/MS. To consider a present peak as a possible interference, the criteria of ± 0.2 min over the retention time of the standard and a signal-to-noise ratio higher than 3 must be taken, according to the criteria in DG-SANCO. LC-QqQ-MS/MS LC Parameters: • Injection volume: 5 μL • Flow rate: 300 μL/min • Column: Zorbax Eclipse PlusC8 1.8 μm, 2.1x100mm • Total run time: 17 + 2.5 min (Post Time) • Mobile Phases and gradient : o A H2O 0.1% formic acid o B AcN 0.1% formic acid and 5% water MS Parameters: • Dynamic MRM • Positive and Negative mode • ESI System: UPLC 1290 coupled to a 6490 Triple Quad LC/MS (Agilent) 0 20 40 60 80 100 120 0 2 15 17 % of mobile phase Time (min) A B Extraction Methods Ethyl Acetate 10 g of Sample + 10 ml of EtOAc Shake 15 min automatically Analysis 1.5 g NaCl + 8 g MgSO 4 Centrifuge 5 minutes at 3700 rpm Centrifuge 5 minutes at 3700 rpm Citrate QuEChERS 10 g of Sample + 10 ml of AcN Analysis 4 g MgSO 4 + 1 g NaCl + 1 g NaAc·2H 2 O +0.5 g NaAc·1.5H 2 O Centrifuge 5 minutes at 3700 rpm Shake 4 min automatically Take 5 mL + 750 mg MgSO4 + 125 mg PSA Shake 4 min automatically Citrate QuEChERS without Clean-up 10 g of Sample + 10 ml of AcN Analysis 4 g MgSO 4 + 1 g NaCl + 1 g NaAc·2H 2 O +0.5 g NaAc·1.5H 2 O Centrifuge 5 minutes at 3700 rpm Shake 4 min automatically Shake 4 min automatically Dutch mini-Luke 15 g of Sample + 20 ml of Acetone + 15 g Na 2 SO 4 Add 20 ml PE + 10 ml DCM Analysis Mix 30 sec by Turrax Centrifuge 3 minutes at 3300 rpm Mix 30 sec by Turrax Conclusions The percentage of compounds showing a interference in the ± 0.2 min retention time range is similar with all the studied methods. Only differences were observed in avocado where the interferences were higher for ethyl acetate and Dutch mimi-Luke extraction methods. Possible interferences detected in LC are much lesser comparing to GC, because of the 5 times dilution factor. Furthermore this fact can be a consequence of the different ionization source used (EI and ESI). The presence of interferences can lead to false negatives/positives and difficulties for a good quantification. But the number of these “potential” bad results can be reduced by decreasing the accepted retention time window for each target compound (e.g. ± 0.1 min). 0 50 100 150 200 250 300 0 5 10 15 20 T (ºC) t (min) GC Parameters: • Injection volume: 2 μL • Splitless mode • Column: HP-5MSUI (15 m x 250 μm x 0.25 μm) • Carrier gas: Helium • Constant pressure: 15.35 psi • Total run time: 20 + 3 min (Post Run) • Oven temperature program: MS Parameters: • MRM • Positive mode • Electron Impact System: 7890 GC System coupled to a 7000 GC/MS Triple Quad (Agilent) GC-QqQ-MS/MS False Negatives Examples Ion Ratio: 0.82 Diff.: 6 % SRM1 (209>133) SRM2 (133>117) Ion Ratio: 0.82 Diff.: 6 % SRM1 (209>133) SRM2 (133>117) Ion Ratio: 1.32 Diff .: 51 % SRM1 (209>133) SRM2 (133>117) Metazachlor at 10 µg/kg in Celery (GC-QqQ-MS/MS) Tomato spiked at 10 µg/L Ethyl Acetate Dutch mini-Luke Citrate QuEChERS SRM1 (209>133) SRM2 (133>117) Ion Ratio: 0.87 Tomato spiked at 10 µg/L Haloxyfop at 10 µg/kg in Aubergine (LC-QqQ-MS/MS) Ion Ratio: 0.38 Ethyl Acetate Ion Ratio: 0.34 Diff.: 11% Citrate QuEChERS Ion Ratio: 0.39 Diff.: 3% Ion Ratio: 0.21 Diff.: 45% Citrate QuEChERS without Clean-up Dutch mini-Luke Ion Ratio: 0.27 Diff.: 29% SRM1 (362>316) SRM2 (362>288) SRM1 (362>316) SRM2 (362>288) SRM1 (362>316) SRM2 (362>288) SRM1 (362>316) SRM2 (362>288) SRM1 (362>316) SRM2 (362>288) Spiking most problematic matrices, the presence of a interference can lead to a wrong identification, due to the influence on the ion ratio, and providing a false negative result. As can be seen in the chromatograms, the interferences can also be related to the extraction method. Amount Injected: o LC: 0.2 g of sample/ml of extract o GC: 1 g of sample/ml of extract 0 10 20 30 40 50 60 70 80 90 100 x < 20 20 < x < 50 x > 50 % of Compounds |Matrix Effect |(%) Leek Linearity and Matrix Effect 0 10 20 30 40 50 60 70 80 90 100 x < 20 20 < x < 50 x > 50 % of Compounds |Matrix Effect| (%) Avocado Citrate QuEChERS with no Clean-up Citrate QuEChERS Ethyl Acetate Dutch mini-Luke 0 10 20 30 40 50 60 70 80 90 100 x < 20 20 < x < 50 x > 50 % of Compounds |Matrix Effect| (%) Broccoli Citrate QuEChERS with no Clean-up Citrate QuEChERS Ethyl Acetate Dutch mini-Luke 0 10 20 30 40 50 60 70 80 90 100 x < 20 20 < x < 50 x > 50 % of Compounds |Matrix Effect| (%) Cucumber 0 10 20 30 40 50 60 70 80 90 100 x < 20 20 < x < 50 x > 50 % of Compounds |Matrix Effect| (%) Leek GC-QqQ/MSMS 0 10 20 30 40 50 60 70 80 90 100 x < 20 20 < x < 50 x > 50 % of Compounds |Matrix Effect| (%) Avocado Citrate QuEChERS with no Clean-up Citrate QuEChERS Ethyl Acetate Dutch mini-Luke 0 10 20 30 40 50 60 70 80 90 100 x < 20 20 < x < 50 x > 50 % of Compounds |Matrix Effect| (%) Broccoli Citrate QuEChERS with no Clean-up Citrate QuEChERS Ethyl Acetate Dutch mini-Luke 0 10 20 30 40 50 60 70 80 90 100 x < 20 20 < x < 50 x > 50 % of Compounds |Matrix Effect |(%) Cucumber LC-QqQ/MSMS The ten most difficult matrix extracts were selected and spiked at four concentration levels for each extraction method, both for LC and GC. From these analysis, linearity and matrix effect were studied. Regards linearity, all matrices/extraction methods showed a good response, with residual values lower than 20 % for each concentration level, and correlation coefficient higher than 0.99. To evaluate matrix effect, slopes obtained for each commodity/extraction method were compared to that one resulting in solvent. In LC, increasing the complexity of the matrix also increase the percentage of compounds showing moderate (20% < x < 50%) or strong (x > 50%) matrix effect, belonging to a suppression phenomenon close to 70 % of results. Contrary to this, in GC a 10 % of compounds as much presented no matrix effect (x < 20%); and, for the rest, close to 98 % of compounds showed enhancement phenomena. 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 0 50 100 150 200 250 Area C (µg/L) Benalaxyl in Avocado Citrate QuEChERS with no Clean-up Citrate QuEChERS Ethyl Acetate Dutch mini-Luke Solvent without Clean-up without Clean-up without Clean-up without Clean-up without Clean-up References: [1] Uclés S., Belmonte N., Mezcua M., Martínez A. B., Martínez-Bueno M. J., Gamón M., Fernández-Alba A. R., Journal of Environmental Science and Health, Part B, 2014, 49, 557-568. [2] Lozano A., de Kok A., Kiedrowska B., Scholten J. and Fernández-Alba A.R., Food Control, 2015, accepted. THE AUTHORS ACKNOWLEDGE FUNDING SUPPORT FROM THE EUROPEAN COMMISSION, DG SANCO, OF THE ANNUAL FINANCING DECISION (2011/889/EU) 0 2 4 6 8 10 12 14 Aubergine Broccoli Green Beans Parsley Leek Cucumber Strawberry Celery Potato Avocado % of Compounds Citrate QuEChERS Ethyl Acetate Dutch mini-Luke Interferences in Selected Reaction Monitoring 0 2 4 6 8 10 12 14 Tomato Aubergine Apple Cauliflower Carrot Broccoli Lemon Green Beans Parsley Lettuce Cherry Leek Cucumber Pepper Srawberry Celery Orange Potato Melon Banana Pumpkin Onion Pear Avocado % of Compounds Ethyl Acetate 2 transitions 1 transition 0 2 4 6 8 10 12 14 Tomato Aubergine Apple Cauliflower Carrot Broccoli Lemon Green Beans Parsley Lettuce Cherry Leek Cucumber Pepper Srawberry Celery Orange Potato Melon Banana Pumpkin Onion Pear Avocado Green Tea % of Compounds Dutch mini-Luke 0 2 4 6 8 10 12 14 Tomato Aubergine Apple Cauliflower Carrot Broccoli Lemon Green Beans Parsley Lettuce Cherry Leek Cucumber Pepper Srawberry Celery Orange Potato Melon Banana Pumpkin Onion Pear Avocado Green Tea % of Compounds Citrate QuEChERS 0 2 4 6 8 10 12 14 Tomato Aubergine Apple Cauliflower Carrot Broccoli Lemon Green Beans Parsley Lettuce Cherry Leek Cucumber Pepper Srawberry Celery Orange Potato Melon Banana Pumpkin Onion Pear Avocado Green Tea % of Compounds Citrate QuEChERS without Clean-up 2 transitions 1 transition 0 2 4 6 8 10 12 14 Tomato Aubergine Apple Cauliflower Carrot Broccoli Lemon Green Beans Parsley Lettuce Cherry Leek Cucumber Pepper Srawberry Celery Orange Potato Melon Banana Pumpkin Onion Pear Avocado % of Compounds Ethyl Acetate 2 transitions 1 transition 0 2 4 6 8 10 12 14 Tomato Aubergine Apple Cauliflower Carrot Broccoli Lemon Green Beans Parsley Lettuce Cherry Leek Cucumber Pepper Srawberry Celery Orange Potato Melon Banana Pumpkin Onion Pear Avocado Green Tea % of Compounds Dutch mini-Luke LC-QqQ/MSMS GC-QqQ/MSMS In the case of LC-QqQ-MS/MS, around 160 compounds have been monitored. There are no differences between the methods tested, never exceeding 3 % of interferences, in the ± 0.2 min retention time range. The matrices presenting the larger number of interferences were green beans, leek and celery. On the other hand, the study developed by GC-QqQ-MS/MS has been carried out monitoring over 220 pesticides. Highest number of interferences can be seen for ethyl acetate extraction method and Dutch mini-Luke method, due to the solvents employed for these extractions are more apolar (ethyl acetate and acetone, respectively), but in any case interferences exceed 15 % (avocado with ethyl acetate method), in the ± 0.2 min retention time range. In all methods the highest number of interferences was obtained for avocado, nevertheless other complex matrices detected were parsley and cucumber. The ten matrices presenting highest percentage of interferences were selected, and their extracts were spiked to study false negatives generated by a matrix peak interference. To develop this study, only interferences overlaping with compound peaks, and so making the difference in ion ratio > 30 %, were taken into account. Again, higher number of false negatives at 10 µg/kg can be observed in results provided by GC-QqQ-MS/MS due to the ionization source employed in GC (EI) generate less selective transitions. To decrease the percentage of false negatives two options can be considered: try to select a third more selective transition and/or confirm with other chromatographic technique. “Potential” False Negatives at 10 µg/kg without further confirmation LC-QqQ-MS/MS GC-QqQ-MS/MS CQ without Clean-up 0 2 4 6 8 10 12 14 Broccoli Lemon Parsley Lettuce Leek Cuccumber Celery Orange Banana Avocado % of Compounds 0 2 4 6 8 10 12 14 Tomato Aubergine Apple Cauliflower Carrot Broccoli Lemon Green Beans Parsley Lettuce Cherry Leek Cucumber Pepper Srawberry Celery Orange Potato Melon Banana Pumpkin Onion Pear Avocado Green Tea % of Compounds Citrate QuEChERS without Clean-up 2 transitions 1 transition 0 2 4 6 8 10 12 14 Tomato Aubergine Apple Cauliflower Carrot Broccoli Lemon Green Beans Parsley Lettuce Cherry Leek Cucumber Pepper Srawberry Celery Orange Potato Melon Banana Pumpkin Onion Pear Avocado Green Tea % of Compounds Citrate QuEChERS
Transcript of Matrix interferences evaluation employing GC and LC ... · PDF fileMatrix interferences...
Matrix interferences evaluation employing GC and LC coupled to triple quadrupole tandem mass spectrometry
Uclés S.1, Lozano A.1, Sosa A.2, Amadeo R. Fernández-Alba1
1 Agrifood Campus of International Excellence (ceiA3), European Union Reference Laboratory for Pesticide Residues in Fruits and Vegetables. University of Almería (Spain); e-mail: [email protected] Agricultural Technology Institute (INTA). Concordia Agricultural Experimental Station (Argentina)
INTRODUCTIONGas and liquid chromatography coupled to triple quadrupole tandem mass spectrometry are two very powerful tools commonly employed for the analysis of pesticide residues in fruit and vegetables. However, due to the big quantity and complexity of the commodities different responses are produced, making difficult to identify/quantify a compound.
To get a clear evaluation twenty five different matrices were purchased from local markets in Almería, extracted with four commonly used extraction methods (citrate QuEChERS with/without clean-up, ethyl acetate extraction method [1], and Dutch mini-Luke method [2]), and evaluated with both GC-QqQ-MS/MS and LC-QqQ-MS/MS. To consider a present peak as a possible interference, the criteria of ± 0.2 min over the retention time of the standard and a signal-to-noise ratio higher than 3 must be taken, according to the criteria in DG-SANCO.
LC-QqQ-MS/MS
LC Parameters: • Injection volume: 5 µL• Flow rate: 300 µL/min• Column: Zorbax Eclipse PlusC8 1.8 μm, 2.1x100mm• Total run time: 17 + 2.5 min (Post Time)• Mobile Phases and gradient :
o A H2O 0.1% formic acido B AcN 0.1% formic acid and 5% water
MS Parameters: • Dynamic MRM• Positive and Negative mode• ESI
System:UPLC 1290 coupled to a 6490 Triple Quad LC/MS (Agilent)
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A
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Extraction MethodsEthyl Acetate
10 g of Sample + 10 ml of EtOAc
Shake 15 min automatically
Analysis
1.5 g NaCl + 8 g MgSO4
Centrifuge 5 minutes at 3700 rpm
Centrifuge 5 minutes at 3700 rpm
Citrate QuEChERS
10 g of Sample + 10 ml of AcN
Analysis
4 g MgSO4 + 1 g NaCl +
1 g NaAc·2H2O +0.5 g NaAc·1.5H2O
Centrifuge 5 minutes at 3700 rpm
Shake 4 min
automatically
Take 5 mL + 750 mg MgSO4 + 125 mg PSA
Shake 4 min
automatically
Citrate QuEChERSwithout Clean-up
10 g of Sample + 10 ml of AcN
Analysis
4 g MgSO4 + 1 g NaCl +
1 g NaAc·2H2O +0.5 g NaAc·1.5H2O
Centrifuge 5 minutes at 3700 rpm
Shake 4 min
automatically
Shake 4 min
automatically
Dutch mini-Luke
15 g of Sample + 20 ml of
Acetone + 15 g Na2SO4
Add 20 ml PE + 10 ml DCM
Analysis
Mix 30 sec by Turrax
Centrifuge 3 minutes at 3300 rpm
Mix 30 sec by Turrax
Conclusions The percentage of compounds showing a interference in the ± 0.2 min retention time range is similar with all the studied methods. Only differences were observed in avocado where the interferences were higher for ethyl acetate and Dutch mimi-Luke extraction methods.
Possible interferences detected in LC are much lesser comparing to GC, because of the 5 times dilution factor. Furthermore this fact can be a consequence of the different ionization source used (EI and ESI).
The presence of interferences can lead to false negatives/positives and difficulties for a good quantification. But the number of these “potential” bad results can be reduced by decreasing the accepted retention time window for each target compound (e.g. ± 0.1 min).
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GC Parameters: • Injection volume: 2 µL• Splitless mode• Column: HP-5MSUI (15 m x 250 µm x 0.25 µm)• Carrier gas: Helium• Constant pressure: 15.35 psi• Total run time: 20 + 3 min (Post Run)• Oven temperature program:
MS Parameters: • MRM• Positive mode• Electron Impact
System:7890 GC System coupled to a 7000 GC/MS Triple Quad (Agilent)
GC-QqQ-MS/MS
False Negatives Examples
Ion Ratio: 0.82Diff.: 6 %
SRM1 (209>133)
SRM2 (133>117)
Ion Ratio: 0.82Diff.: 6 %
SRM1 (209>133)
SRM2 (133>117)Ion Ratio: 1.32Diff .: 51 %
SRM1 (209>133)
SRM2 (133>117)
Metazachlor at 10 µg/kg in Celery (GC-QqQ-MS/MS)
Tomato spiked at 10 µg/L
Ethyl Acetate Dutch mini-LukeCitrate QuEChERS
SRM1 (209>133)
SRM2 (133>117)
Ion Ratio: 0.87
Tomato spiked at 10 µg/L
Haloxyfop at 10 µg/kg in Aubergine (LC-QqQ-MS/MS)
Ion Ratio: 0.38
Ethyl Acetate
Ion Ratio: 0.34Diff.: 11%
Citrate QuEChERS
Ion Ratio: 0.39Diff.: 3%
Ion Ratio: 0.21Diff.: 45%
Citrate QuEChERSwithout Clean-up
Dutch mini-Luke
Ion Ratio: 0.27Diff.: 29%
SRM1 (362>316)
SRM2 (362>288)
SRM1 (362>316)
SRM2 (362>288)
SRM1 (362>316)
SRM2 (362>288)
SRM1 (362>316)
SRM2 (362>288)
SRM1 (362>316)
SRM2 (362>288)
Spiking most problematic matrices, the presence of a interference can lead to a wrong identification, due to the influence on the ion ratio, and providing a false negative result.
As can be seen in the chromatograms, the interferences can also be related to the extraction method.
Amount Injected:
o LC: 0.2 g of sample/ml of extract
o GC: 1 g of sample/ml of extract
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poun
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|Matrix Effect |(%)
Leek
Linearity and Matrix Effect
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|Matrix Effect| (%)
Avocado
Citrate QuEChERS with no Clean-up Citrate QuEChERS Ethyl Acetate Dutch mini-Luke
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x < 20 20 < x < 50 x > 50
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|Matrix Effect| (%)
Broccoli
Citrate QuEChERS with no Clean-up Citrate QuEChERS Ethyl Acetate Dutch mini-Luke
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Cucumber
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Avocado
Citrate QuEChERS with no Clean-up Citrate QuEChERS Ethyl Acetate Dutch mini-Luke
0102030405060708090
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Broccoli
Citrate QuEChERS with no Clean-up Citrate QuEChERS Ethyl Acetate Dutch mini-Luke
0102030405060708090
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Cucumber
LC-QqQ/MSMS
The ten most difficult matrix extracts were selected and spiked at four concentration levels for each extraction method, both for LC and GC. From these analysis, linearity and matrix effect were studied.
Regards linearity, all matrices/extraction methods showed a good response, with residual values lower than 20 % for each concentration level, and correlation coefficient higher than 0.99.
To evaluate matrix effect, slopes obtained for each commodity/extraction method were compared to that one resulting in solvent. In LC, increasing the complexity of the matrix also increase the percentage of compounds showing moderate (20% < x < 50%) or strong (x > 50%) matrix effect, belonging to a suppression phenomenon close to 70 % of results. Contrary to this, in GC a 10 % of compounds as much presented no matrix effect (x < 20%); and, for the rest, close to 98 % of compounds showed enhancement phenomena.
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C (µg/L)
Benalaxyl in Avocado
Citrate QuEChERS with no Clean-up Citrate QuEChERS Ethyl Acetate Dutch mini-Luke Solvent
without Clean-up without Clean-upwithout Clean-upwithout Clean-up
without Clean-up
References:[1] Uclés S., Belmonte N., Mezcua M., Martínez A. B., Martínez-Bueno M. J., Gamón M., Fernández-Alba A. R., Journal of Environmental Science and Health, Part B, 2014, 49, 557-568. [2] Lozano A., de Kok A., Kiedrowska B., Scholten J. and Fernández-Alba A.R., Food Control, 2015, accepted.
THE AUTHORS ACKNOWLEDGE FUNDING SUPPORT FROM THE EUROPEAN COMMISSION, DG SANCO, OF THE ANNUAL FINANCING DECISION (2011/889/EU)
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Interferences in Selected Reaction Monitoring
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LC-QqQ/MSMS GC-QqQ/MSMS
In the case of LC-QqQ-MS/MS, around 160 compounds have been monitored. There are no differences between the methods tested, never exceeding 3 % of interferences, in the ± 0.2 min retention time range. The matrices presenting the larger number of interferences were green beans, leek and celery.
On the other hand, the study developed by GC-QqQ-MS/MS has been carried out monitoring over 220 pesticides. Highest number of interferences can be seen for ethyl acetate extraction method and Dutch mini-Luke method, due to the solvents employed for these extractions are more apolar (ethyl acetate and acetone, respectively), but in any case interferences exceed 15 % (avocado with ethyl acetate method), in the ± 0.2 min retention time range. In all methods the highest number of interferences was obtained for avocado, nevertheless other complex matrices detected were parsley and cucumber.
The ten matrices presenting highest percentage of interferences were selected, and their extracts were spiked to study false negatives generated by a matrix peak interference. To develop this study, only interferences overlaping with compound peaks, and so making the difference in ion ratio > 30 %, were taken into account. Again, higher number of false negatives at 10 µg/kg can be observed in results provided by GC-QqQ-MS/MS due to the ionization source employed in GC (EI) generate less selective transitions. To decrease the percentage of false negatives two options can be considered: try to select a third more selective transition and/or confirm with other chromatographic technique.
“Potential” False Negatives at 10 µg/kg without further confirmation
LC-QqQ-MS/MS GC-QqQ-MS/MS
CQ without Clean-up
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eCh
erry
Leek
Cucu
mbe
rPe
pper
Sraw
berr
yCe
lery
Ora
nge
Pota
toM
elon
Bana
naPu
mpk
inO
nion
Pear
Avo
cado
Gre
en T
ea
% o
f Com
poun
ds
Citrate QuEChERS without Clean-up
2 transitions
1 transition
0
2
4
6
8
10
12
14
Tom
ato
Aub
ergi
neA
pple
Caul
iflow
erCa
rrot
Broc
coli
Lem
onG
reen
Bea
nsPa
rsle
yLe
ttuc
eCh
erry
Leek
Cucu
mbe
rPe
pper
Sraw
berr
yCe
lery
Ora
nge
Pota
toM
elon
Bana
naPu
mpk
inO
nion
Pear
Avo
cado
Gre
en T
ea
% o
f Com
poun
ds
Citrate QuEChERS
